This blog is meant to allow Fragment-based Drug Design Practitioners to get together and discuss NON-CONFIDENTIAL issues regarding fragments.

24 April 2017

Fragment optimization without purification

Compound purification can be a major hassle: separating the
desired product from starting materials, reagents, and byproducts often takes
far longer than making the compound in the first place. As we’ve previously
noted, this is especially true for small, polar fragments – which are particularly
attractive for drugs. Two new papers address this challenge. (Shameless plug: my company Carmot Therapeutics also has a solution to this problem.)

In J. Med. Chem.,
Paul Brough and Vernalis colleagues describe their discovery of inhibitors of
all four isoforms of pyruvate dehydrogenase kinase (PDHK), potential targets
for diabetes and oncology. The ATP-binding site of these four enzymes is
similar to that of oncology target HSP90, in which Vernalis has a long-standing
interest.

A screen of 1063 fragments (each at 0.5 mM) against PDHK-2 yielded
78 hits that were positive in three different NMR-based assays and also
ATP-competitive. These yielded a whopping 43 structures when soaked into
crystals of the related isoform PDHK-3. Compound 6 was one, and the binding mode
was very similar to that previously seen for the same fragment with HSP90.
Fragment growing rapidly led to molecules such as compound 8, with low
micromolar potency. This compound was almost equipotent against HSP90, but
modeling suggested that it might be possible to further grow this molecule in a
direction that would be accommodated in the PDHKs but not in HSP90.

The next step was to make a bunch of analogs, and here's where avoiding purification becomes advantageous. Specifically, the researchers turned
to off-rate screening (ORS), which entails
making compounds and then testing the impure mixtures using surface plasmon
resonance (SPR) to look for those which dissociate more slowly. Since off-rate
is not dependent on the concentration of ligand, a low yield shouldn’t change
the results of the assay.

An initial library of 56 compounds led to the discovery of
compound 18, and subsequent libraries and medicinal chemistry ultimately
yielded VER-246608, which is a potent pan-PDHK inhibitor. As designed, it
is also completely inactive against HSP90. The molecule is described more
thoroughly in this Oncotargetpaper,
which reveals that despite activity against PDHKs in cells, VER-246608 is not
particularly effective at slowing the proliferation of cancer cells. Still, it
does appear to be a useful chemical probe for further exploring the biology of
the PDHKs.

Shifting methods but staying with the theme of assaying
impure compounds brings us to a paper in SLAS
Discovery by Sten Ohlson, Brian Dymock, and colleagues at Nanyang
Technological University and the National University of Singapore. The protein
tested was HSP90, and the method used was weak affinity chromatography, or WAC
(see here, here, and here).

Like SPR, WAC also uses an immobilized protein. However,
whereas SPR provides the (kinetic) off-rate, WAC provides the (thermodynamic)
dissociation constant, which is calculated from the change in retention time of
the molecule as it passes through a column containing protein-bound resin. In
this case the researchers synthesized a mixture of five different compounds
which varied from 7-24% of the mixture. This crude sample was analyzed by WAC,
and the resulting dissociation constants, ranging from 48-147 µM, were
satisfactorily similar to the values obtained using pure compounds.

Both of these approaches should accelerate screening and
facilitate the analysis of complicated mixtures, such as natural product extracts.
It will be fun to watch for more examples.